Serological diagnostic testing is of growing importance in the early detection and differentiation of rheumatoid arthritis (RA). Apart from the traditional detection of the rheumatoid factor, new specific autoantibodies to citrullinated antigens have made a crucial contribution to the diagnosis of RA.[26]
The rheumatoid factor is an autoantibody, which may be IgM, IgG or IgA, and first mentioned in 1922.[1] It recognizes domains CH2 and CH3 of the Fc segment of human IgG and is a component of the classification criteria for RA published by the American College of Rheumatology.[2]
Rheumatoid factor (RF) can be determined by various test methods; ELISA (enzyme-linked immunosorbent assay) and nephelometry are standardized methods. The mean specificity of detection of RF is about 79%, with sensitivity of about 60%.[3] In recent years, new autoantibodies have been detected and characterized in the sera of RA patients. These exhibit higher specificity and can, therefore, help to improve serological diagnostic testing.
One of the most important serological discoveries in rheumatology in recent years has been the characterization of autoantibodies directed against citrullinated filaggrin.[4] The starting point for this discovery was the identification of the target antigen for anti-keratin antibodies (AKA). These were first described in 1979 and are highly specific for RA.[4]
The target antigen filaggrin is only expressed in epithelial cells and not in joints or other organs; the pathogenetic significance of this finding was, therefore, initially unclear. Schellekens et al.[6] showed that only citrullinated forms of filaggrin were recognized by AKA.
Filaggrin may be citrullinated by enzymatic deimination of arginine residues to give citrulline residues. Citrullination is a post-translational modification, which alters the charge of a protein, leading to changes in its three-dimensional structure, which, in turn, results in changes in antigenic properties.[7] Citrullination has an essential physiological and biochemical role in cell differentiation and in programmed cell death (apoptosis).
First generation ELISAs for the detection of anti-citrullinated filaggrin showed a specificity for RA of about 85%, with sensitivity of 65% to 70%. Second generation ELISAs used synthetic peptides as antigen, with a ring structure due to intramolecular disulfide bridge formation. Use of these cyclic citrullinated peptides (CCP) has improved the specificity to between 96% and 98%, without changing the sensitivity.[8]
Several studies have now shown that anti-CCP antibodies are not only highly specific, but also of high predictive value for an erosive course of the disease and are thus of prognostic value.[9]
Studies with citrullinated peptide derivatives of fibrin and fibrinogen have now shown a cross-reaction between filaggrin and citrullinated fibrin.[12] Some publications have found high diagnostic specificity and sensitivity for the detection of anti-citrullinated fibrinogen antibodies in patients with RA.[13] With ELISA, the sensitivity for RA was about 75%, with specificity of 98%.
Another interesting citrullinated autoantigen is the citrullinated form of alpha-enolase, an enzyme that plays a role in glycolysis.[15] Citrullinated alpha-enolase has been detected—together with other citrullinated antigens—in the synovial tissue of patients with rheumatoid arthritis.[16]
Citrullinated vimentin has been described as a relevant autoantigen expressed in synovial tissue. Subsequently, it was clarified that citrullinated vimentin is identical to the formerly known antigen Sa, which stands for Savoie, the name of the patient in whom the respective autoantibody response was first identified. Anti-Sa antibodies provide a high specificity of >98%, but a limited sensitivity of 22% to 40% for patients with rheumatoid arthritis.[17]
An ELISA based on mutated citrullinated vimentin (MCV) has been commercially available for the diagnosis of rheumatoid arthritis for some time and has about the same diagnostic sensitivity and specificity as anti-CCP antibodies.[18, 19, 20]
Antibodies against carbamylated proteins (proteins comprising a homo-citrulline residue) have also been described to occur in the serum of patients with RA.[28, 29] Homocitrulline was found to be present in rheumatoid nodule, together with citrulline,[28] and antibodies against carbamylated proteins were found to predict joint damage.[29]
Two serological point-of-care tests (POCT) for the early detection of RA have been very recently developed. The Rheuma-Chec test (Orgentec, Mainz, Germany) combines two biomarkers for the diagnosis of RA—rheumatoid factor and antibodies to MCV. Antibodies to CCP are detected with the CCPOINT™ assay (Euro-Diagnostica, Malmö, Sweden).[25] The tests require only a single drop of whole blood and any general physician can perform them within minutes.
Despite the above advances, there is still room for improvement of the existing serological tests for rheumatoid arthritis, in particular, with respect to sensitivity, specificity and ease of handling.